The PIWI interaction RNA (piRNA) pathway is required for transposon silencing during germline development in flies, fish and mouse. The 24 to 29 nucleotide long piRNAs are produced by a Dicer-independent mechanism and associate specifically with PIWI class Argonaute proteins. These piRNA-PIWI complexes can cleave target RNAs in vitro. However, the in vivo mechanism of transposon silencing and the genomic origins of piRNAs are not understood. In addition, the full spectrum of biological functions for the piRNA pathway remains to be explored. These broad questions will be addressed using a combination of genetic, genomic, cytological and molecular approaches in the experimentally tractable Drosophila system. The broad aims of the proposal are to define the structural and biochemical properties of piRNA encoding clusters, determine the mechanism of transposon silencing by piRNAs, and to characterize recently identified functions for this pathway in meiotic and mitotic chromosome segregation. Studies under aim 1 will use computational analysis of deep sequencing data and chromatin immunoprecipitation to define properties of piRNA encoding clusters. Aim 2 will use transgenic reporters to explore the mechanism of piRNA silencing. Studies under Aim 3 will use genetic, molecular and cytological approaches to define the role of piRNA in telomere protection and chromosome segregation.